title: Outer membrane phospholipase a dimer stability does not correlate to occluded surface area.
abstract: Despite the key roles of oligomeric membrane proteins (MPs) in many known cellular pathways, the principles governing their oligomer stability are not well-understood. Previous work with the alpha-helical MPs bacteriorhodopsin (bR) and glycophorin A (GpA) shows that lost buried surface area linearly correlated with perturbations in protein stability. Although this is a significant discovery, the predictive power of this correlation is limited by the data. Because both bR and GpA have alpha-helical secondary structural motifs, it is unclear whether this correlation would be observed for MPs with a beta-barrel motif. We addressed this question by measuring the thermodynamic consequences of interfacial amino acid changes at the dimer interface of beta-barrel MP outer membrane phospholipase A (OMPLA). We created sequence variants to reduce the contact surface area of the OMPLA dimer interface by introducing single-alanine substitutions and used sedimentation equilibrium analytical ultracentrifugation to determine the dimerization free energies for these variants. The integrity of each variant was verified by two functional assays: specific activity and resistance to thermal denaturation, which showed that structural changes were restricted to the local environment. Using this information, we calculated the anticipated packing defects due to side chain deletion and compared this to the free energy perturbations for each residue. Contrary to the findings with bR and GpA, our study found no correlation between the contact surface area lost and the perturbations to OMPLA dimer stability. We conclude that van der Waals packing may not be a strong predictor of stability for all membrane proteins.
authors.affiliation: T. C. Jenkins Department of Biophysics, Johns Hopkins University, 3400 North Charles Street, Baltimore, Maryland 21218, USA. Karen.Fleming@jhu.edu
authors.forename: Alexandra
authors.initials: A
authors.lastname: Ebie Tan
authors.forename: Karen G
authors.initials: KG
authors.lastname: Fleming
mesh_terms: D020816:Amino Acid Motifs / Q000502:physiology; D019943:Amino Acid Substitution; D001425:Bacterial Outer Membrane Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D019281:Dimerization; D004795:Enzyme Stability / Q000502:physiology; D004926:Escherichia coli / Q000201:enzymology* / Q000235:genetics; D020125:Mutation, Missense; D054466:Phospholipases A1 / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D020836:Protein Structure, Quaternary / Q000502:physiology; D013499:Surface Properties
chemical_list: D001425:Bacterial Outer Membrane Proteins; D054466:Phospholipases A1; C412876:outer membrane phospholipase A